T lymphocyte maturation in the thymus is maintained by the continuous influx of hematopoietic progenitors that are ultimately derived from bone marrow hematopoietic stem cells. Transcription factor GATA-3 is a critical regulator of T cell and thymic natural killer (NK) cell development, and has been shown to be vital for multiple stages of T cell development. Our recent studies revealed that GATA-3 is required for the development of the earliest T cell progenitor (ETP), the most immature T cell in the thymus. By way of contrast, we also demonstrated that GATA-3 is not required prior to the ETP stage for the development of fetal liver or adult bone marrow pre-thymic progenitors that bear T cell potential. This vital demonstration was functionally difficult to address because no T cells can be generated from GATA3-null progenitors, but we were able to definitively demonstrate this requirement using a novel Gata3-eGFP hypomorphic allele. Thus GATA-3 is required at the earliest stage of T lymphopoiesis as well as at intermediate and late stages of thymocyte development and for CD4+ T cell Th2 differentiation in the periphery. Although the transcriptional hierarchy mediated by GATA-3 (the transcription factor) and through Gata3 (the gene) is beginning to be characterized in peripheral T cells, its hierarchical activity in the T cell transcriptional regulaory network in the thymus remains largely unknown. We recently identified a Gata3 T and NK cell-specific enhancer located 280 kbp 3' to the Gata3 structural gene. Here we propose to characterize the GATA-3-centric transcriptional network that controls T cell development from the most immature hematopoietic progenitor stages through to the mature Th2 stage. Contributing to our understanding of GATA-3 regulation in early thymopoiesis may be critical for eventually deciphering the etiology and progression of T cell leukemias and lymphomas, since aberrant GATA- 3 expression has been implicated as a causal agent in a subset of these diseases. PUBLIC HEALTH RELEVANCE: T lymphocytes are an indispensable component of the vertebrate adaptive immune response, and defects or aberrant development during T lymphopoiesis can result in compromised immunological competence or leukemia. The interplay of multiple transcription factors regulates proper T lymphocyte development, and GATA-3 plays a critical role in their maturation and activation. Successful execution of this research program will fundamentally contribute to our understanding of the transcriptional network(s) that controls T cell development from hematopoietic stem cells, which in turn should contribute in the future to the development of new therapeutic approaches to hematopoietic diseases, including leukemia.